PROJECT SUMMARY Chemotherapy continues to be an important component of therapy for breast cancer, given to reduce the risk of metastatic recurrence. Although response (sensitivity) or lack of response (resistance) to chemotherapy is associated with prognosis, there is currently no validated commercial assay available which specifically predicts response to chemotherapy. Personalized approaches to cancer therapy are needed to select a drug or combination of drugs to which a tumor is most sensitive, and to avoid the toxicity of drugs to which the tumor is or becomes resistant. Such biomarker will assist oncologists in the daily clinical management of aging patients who are fragile to multiple chemotherapies and triple negative breast cancer patients whose major treatment option is chemotherapy. Coactivator associated arginine methyltransferase 1 (CARM1) is a protein arginine (R) methyltransferase which can methylate histone H3 and a variety of non-histone substrates. We recently identified a mediator of RNA polymerase II transcription subunit 12 (MED12) as a novel substrate for CARM1. The proposed project will determine whether methylation of MED12 is a predictor for chemo-sensitivity in breast cancer. We have mapped the methylation sites of MED12 to R1862 and R1912. Coincidently, mutations on R1862 had been reported in lung and cervical cancers, and mutations on R1912 had been found in a melanoma patient who developed resistance to BRAF inhibitor. We found that overexpression of MED12 wild-type, but not MED12R1862K,R1912K mutant, increased sensitivity of HEK293 cells to 5-FU and anthracyclines. Interestingly, the MED12 methylation dependent mechanism is distinct from activation of TGF-?R signaling as reported for complete knockout of MED12 in lung and colon cancers. Further, we have identified targets regulated by methylated MED12 that may determine chemo-sensitivity. We hypothesize that methylation of MED12 by CARM1 represents an important mechanism conferring chemosensitivity. The proposed research will directly address (1) whether MED12 methylation predicts sensitivity of breast cancer cells to commonly used chemotherapies in cell culture and xenograft tumor models; (2) define the mechanism of methylated-MED12 controlled chemosensitivity; and (3) test the clinical relevance of the MED12 methylation in chemo- resistance using large cohorts of clinical specimens. The goal is to delineate the methylation dependent mechanism for chemotherapy resistance and uncover new targets for therapeutic intervention.